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. 1999 Jul 6;96(14):7791-6.
doi: 10.1073/pnas.96.14.7791.

Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription

P D Bieniasz  1 , T A Grdina, H P Bogerd, B R Cullen
Affiliations

Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription

P D Bieniasz et al. Proc Natl Acad Sci U S A. .

Abstract

Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle. Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of positive transcription elongation factor b (P-TEFb) to TAR. Here, we demonstrate that both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is artificially recruited to a heterologous promoter proximal RNA target. The level of activation observed was indistinguishable from the level induced by Tat and was neither inhibited nor increased when Tat was expressed in trans. Activation by artificially recruited CycT1 depended on the ability to bind the CDK9 component of P-TEFb. In contrast, although binding to both Tat and TAR was essential for the ability of CycT1 to act as a Tat cofactor, these interactions became dispensable when CycT1 was directly recruited to the LTR. Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation. Together, these data demonstrate that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat/TAR axis in viral transcription is to permit the recruitment of CycT1/P-TEFb.

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Figures

Figure 1
Figure 1
Recruitment of CycT1/P-TEFb activates the HIV-1 LTR promoter independently of Tat and TAR. 293T cells were transfected with HIV-1 LTR-based reporter plasmids containing the TAR (a) or SLIIB (b) RNA target. Transactivation by the indicated effectors, expressed either as wild-type unfused proteins or fused to Rev, was determined by measuring CAT levels 48 hr after transfection. In c, cells were transfected with the pHIV/SLIIB/CAT reporter plasmid, along with plasmids expressing hCycT1-Rev and the indicated wild-type or mutant Tat expression plasmid. In each case, results represent the mean ± standard deviation of three transfections.
Figure 2
Figure 2
hCycT1-Rev forms a complex with Tat that activates the HIV-1 LTR equivalently via TAR or SLIIB. Murine LmTK-cells were transfected with HIV-1 LTR-based reporter plasmids containing TAR (a) or SLIIB (b) RNA target elements, as in Fig. 1, and plasmids expressing Rev or hCycT1-Rev, along with the indicated Tat expression plasmid. The depicted results reflect mean CAT levels determined 48 hr after transfection ± the standard deviation of three transfection experiments.
Figure 3
Figure 3
Tat and TAR binding by hCycT1 is dispensable for activation when P-TEFb is artificially recruited to the HIV-1 LTR. (a) Analysis of hCycT1 interactions with CDK9, Tat, and TAR in yeast cells. Interactions between VP16-hCycT1 fusion proteins and GAL4-CDK9 or GAL4-Tat were measured by two-hybrid assay. Alternatively, the ability of hCycT1/Tat complexes to interact with HIV-1 TAR was determined by yeast three-hybrid assay. Values indicate the level of β-galactosidase activity in yeast cells after growth on appropriate selective media. (b) The ability of CycT1 to interact with Tat, TAR, and CDK9 is required to support Tat/TAR function. Murine LmTK-cells were transfected with the pHIV/TAR/CAT reporter plasmid along with plasmids expressing Tat and wild-type or mutant hCycT1. (c) CDK9 but not Tat/TAR-binding activity is required for hCycT1-Rev/SLIIB-mediated activation of the HIV-1 LTR. 293T cells were transfected with the pHIV/SLIIB/CAT reporter along with plasmids expressing the indicated hCycT1-Rev fusion proteins. Values are the mean ± standard deviation of CAT levels determined 48 hr after transfection from three transfections. (d) Relative levels of expression of the indicated Rev fusion proteins in transfected 293T cells were determined by Western blot analysis by using a polyclonal anti-Rev antiserum.
Figure 4
Figure 4
Recruitment of hCycT1 to the HIV-1 LTR promoter enhances transcriptional processivity. (a) Schematic representation of the pHIV/SLIIB/CAT reporter plasmid indicating the positions of the PV UTR and Dist probes used to measure the levels of promoter proximal (T1), intermediate (T2), and distal (Dist) RNA. (b) Ribonuclease protection assay performed by using total RNA extracted from 293T cells transfected with pHIV/SLIIB/CAT and a plasmid expressing Rev (lane 1), Tat-Rev (lane 2), hCycT1-Rev (lane 3), hCycT1(X5)-Rev (lane 4), or hCycT1(K26)-Rev (lane 5). A mock protection assay (lane 6) used RNA from untransfected 293T cells.
Figure 5
Figure 5
Schematic representation of three modes of recruitment of CycT1/P-TEFb to the HIV-1 LTR promoter. As shown in this manuscript, each mode produces an equivalent level of activation of this promoter (see text for detailed discussion).
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